Synergistic biocide composition

ABSTRACT

A biocide composition is provided as an addition to substances that can be infected by harmful microorganisms, in which the biocide composition has at least two active biocidal substances, one of which is 2-methylisothiazolin-3-one. The composition is characterized in that it contains 1,2-benzisothiazolin-3-one, compositions containing 5-chloro-2-methylisothiazolin-3-one being excluded. In comparison with its individual components, the composition of the invention has a synergistic biocidal activity.

This application is a 371 of PCT/EP98/05310 Aug. 20, 1998.

The invention relates to a biocide composition as an addition tosubstances that can be infected by harmful microorganisms. In particularthe invention concerns a biocide composition having at least two activebiocidal substances that interact synergistically, in which one of theactive substances is 2-methylisothiazolin-3-one.

Biocidal agents are used in many fields, for example, to control harmfulbacteria, fungi, or algae. The use of 4-isothiazolin-3-ones (which arealso designated as 3-isothiazolones) in such compositions has been knownfor a long time, since these include very effective biocidal compounds.

One of these compounds is 5-chloro-2-methylisothiazolin-3-one. Whereasit does indeed exhibit good biocidal activity, there are variousdisadvantages when it is handled in practice. For example, the compoundfrequently triggers allergies in persons that use it. Also, there arelegal restrictions in many countries on the AOX value of industrialwaste waters, i.e., a certain concentration of organic chlorine,bromine, and iodine compounds that can adsorb to activated carbon mustnot be exceeded in the water. This prevents the use of5-chloro-2-methylisothiazolin-3-one to the desired extent. Furthermore,the stability of this compound is inadequate in certain circumstances,e.g., at high pH values or in the presence of nucleophiles or reducingagents.

Another known isothiazolin-3-one with biocidal activity is2-methylisothiazolin-3-one. While it is true that the compound avoidsvarious disadvantages of 5-chloro-2-methylisothiazolin-3-one, forexample, the high risk of allergy, its biocidal activity is considerablylower. Thus, a simple substitution of 2-methylisothiazolin-3-one for5-chloro-2-methylisothiazolin-3-one is not possible.

The use of combinations of various isothiazolin-3-ones or combinationsof at least one isothiazolin-3-one and other compounds is also alreadyknown. For example, a synergistic biocidal composition that contains2-methylisothiazolin-3-one (2-methyl-3-isothiazolone) and2-n-octylisothiazolin-3-one (2-n-octyl-3-isothiazolone) is described inEP 0676140 A1.

Synergistic biocide compositions that are combinations of1,2-benzisothiazolin-3-one and an iodopropargyl compound (iodopropinylcompound) are known from U.S. Pat. No. 5,328,926. For example,3-Iodopropargyl-N-butyl carbamate is named as such a compound. In thesaid document, however, no biocide compositions are described thatcontain further active biocidal substances besides1,2-benzisothiazolin-3-one and 3-iodopropargyl-N-butyl carbamate.

In JP 01224306 (Chemical Abstracts, Vol. 112, Nos. 11, Mar. 12, 1990,Abstract No. 93924), a biocide composition is described that consists of2-methylisothiazolin-3-one, 1,2-benzisothiazolin-3-one, and5-chloro-2-methylisothiazolin-3-one.

JP 06092806 (Chemical Abstracts, Vol. 121, Nos. 11, Sep. 12, 1994,Abstract No. 127844) relates to biocide compositions that contain anisothiazolinone, 1,2-benzisothiazolin-3-one, and propanol or a propanolderivative. 2-Methylisothiazolin-3-one is named, for example, as theisothiazolinone and 2-bromo-2-nitropropane-1,3-diol, for example, as thepropanol derivative. However, no reference is made to a composition thatspecifically contains 2-methylisothiazolin-3-one,1,2-benzisothiazolin-3-one, and 2-bromo-2-nitropropane-1,3-diol and issimultaneously free of 5-chloro-2-methylisothiazolin-3-one.

The object of the invention is to provide a biocide composition that isimproved in that its components interact synergistically and thereforecan be used in lower concentrations when used simultaneously, comparedto the concentrations necessary in the case of the individualcomponents. Thus, humans and the environment are exposed to lesspollution and the costs of controlling harmful microorganisms arereduced.

This object is achieved by the invention by means of a biocidecomposition having at least two active biocidal substances, one of whichis 2-methylisothiazolin-3-one. The composition is characterized in thatit contains as a further active biocidal substance1,2-benzisothiazolin-3-one, biocide compositions containing5-chloro-2-methyl-isothiazolin-3-one, being excluded.

The biocide composition of the invention has the advantage that it canreplace active substances used until now in practice, but suffering fromdisadvantages with respect to health and the environment, e.g.,5-chloro-2-methylisothiazolin-3-one. Moreover, the biocide compositionof the invention can be produced with water as a favorable medium, ifnecessary. The addition of emulsifiers, organic solvents, and/orstabilizers is thus not necessary. Moreover the invention makes itpossible to match the composition to specific goals by adding furtheractive substances, for example, in the sense of an increased biocidalactivity, improved long-term protection of the substances infected bymicroorganisms, improved compatibility with the substances to beprotected, or improved toxicological or ecotoxicological behavior.

The biocide composition of the invention contains2-methylisothiazolin-3-one and 1,2-benzisothiazolin-3-one, normally in aweight ratio of (50-1): (1-50), preferably in a weight ratio of (15-1):(1-8), in particular in a weight ratio of (4-1) (1-4). A weight ratio of1:1 is particularly preferred.

In the biocide composition the 2-methylisothiazolin-3-one and the1,2-benzisothiazolin-3-one are present in a total concentration ofpreferably 0.5 to 50% by wt, in particular 1 to 20% by wt, particularlypreferred 2.5 to 10% by wt, in each case relative to the total biocidecomposition.

It is advisable to use the biocides of the composition of the inventionin combination with a polar or nonpolar liquid medium. This medium canbe supplied, for example, in the biocide composition and/or in thesubstance to be preserved.

Preferred polar liquid media are water, an aliphatic alcohol having 1 to4 carbon atoms, e.g., ethanol and isopropanol, a glycol, e.g., ethyleneglycol, diethylene glycol, 1,2-propylene glycol, dipropylene glycol, andtripropylene glycol, a glycol ether, e.g., butyl glycol and butyldiglycol, a glycol ester, e.g., butyl diglycol acetate or2,2,4-trimethylpentanediol monoisobutyrate, a polyethylene glycol, apolypropylene glycol, N,N-dimethylformnamide, or a mixture of suchsubstances. The polar liquid medium is in particular water, where the pHvalue of the corresponding biocide composition is preferably adjusted tobe neutral or weakly alkaline, for example, to a pH value of 7 to 9.Thus, the 2-methylisothiazolin-3-one is then present in dissolved formand the 1,2-benzisothiazolin-3-one is present in finely dispersed form,or both active substances are dissolved.

For example, aromatics, preferably xylene and toluene, serve as nonpolarliquid media.

The biocide composition of the invention can also be combinedsimultaneously with a polar and a nonpolar liquid medium.

In addition to 2-methylisothiazolin-3-one and1,2-benzisothiazolin-3-one, the biocide composition of the invention cancontain one or more other active biocidal substances, selected inaccordance with the field of application. Specific examples of suchadditional active biocidal substances are shown below:

benzyl alcohol

2,4-dichlorobenzyl alcohol

2-phenoxyethanol

2-phenoxyethanol hemiformal

phenylethyl alcohol

5-bromo-5-nitro- 1,3-dioxane

formaldehyde and formaldehyde source materials

dimethyloldimethyl hydantoin

glyoxal

glutardialdehyde

sorbic acid

benzoic acid

salicylic acid

p-hydroxybenzoic acid ester

chloroacetamide

N-methylolchloroacetamide

phenols, such as p-chloro-m-cresol and o-phenylphenol

N-methylolurea

N,N′-dimethylolurea

benzyl formal

4,4-dimethyl- 1,3-oxazolidine

1,3,5-hexahydrotriazine derivatives

quaternary ammonium compounds, such as

N-alkyl-N,N-dimethylbenzyl ammonium chloride and

di-n-decyldimethyl ammonium chloride

cetylpyridinium chloride

diguanidine

polybiguanide

chlorohexidine

1,2-dibromo-2,4-dicyanobutane

3,5-dichloro4-hydroxybenzaldehyde

ethylene glycol hemiformal

tetra(hydroxymethyl)phosphonium salts

dichlorophen

2,2-dibromo-3-nitrilopropionic acid amide

3-iodo-2-propinyl-N-butyl carbamate

methyl-N-benzimidazol-2-yl carbamate

2-n-octylisothiazolin-3-one

4, 5-dichloro-2-n-octylisothiazolin-3-one

4,5-trimethylene-2-methylisothiazolin-3 -one

2,2′-dithiodibenzoic acid di-N-methylamide

benzisothiazolinone derivatives

2-thiocyanomethylthiobenzothiazole

C formals, such as

2-hydroxymethyl-2-nitro- 1,3-propanediol

2-bromo-2-nitropropane- 1,3-diol

methylene bisthiocyanate

reaction products of allantoin

3-Iodo-2-propinyl-N-butyl carbamate, 2-n-octylisothiazolin-3-one,formaldehyde or a formaldehyde source material, and²-bromo-2-nitropropane-1,3-diol are preferred as such other activebiocidal substances.

Examples of the formaldehyde source material are N-formals such as

N,N′-dimethylolurea

N-methylolurea

dimethyloldimethyl hydantoin

N-methylolchloroacetamide

reaction products of allantoin

glycol formals such as

ethylene glycol formal

butyl diglycol formal

benzyl formal

The biocide composition of the invention can also contain othercustomary constituents known as additives to those skilled in the art inthe field of biocides. These are, e.g., thickening agents, defoamingagents, substances to adjust the pH value, perfumes, dispersing agents,and coloring substances.

2-Methylisothiazolin-3-one and 1,2-benzisothiazolin-3-one are knownsubstances. 2-Methylisothiazolin-3-one can be produced, for example,according to U.S. Pat. No. 5,466,818. The reaction product obtainedthereby can be purified, e.g., by column chromatography.

1,2-Benzisothiazolin-3-one is commercially available, for example, underthe trade name Acticide® BW 20 and Acticide® BIT from the company ThorChemie GmbH.

3-Iodo-2-propinyl-N-butyl carbamate is likewise commercially available,for example, from the Troy Chemical Company under the trade namePolyphase®, Polyphase® AF-1, and Polyphase® NP-1, or from OlinCorporation under the trade name Omacide® IPBC 100.

2-n-Octylisothiazolin-3-one is also commercially available, for example,from the company Thor Chemie GmbH under the trade name Acticide® OIT.

Finally 2-bromo-2-nitropropane-1,3-diol is commercially available, forexample, from the company Boots under the trade name Myacide® AS.

According to a first embodiment of the invention, the biocidecomposition of the invention is a system in which the combination of2-methylisothiazolin-3-one and 1,2-benzisothiazolin-3-onesynergistically develops a biocidal activity greater than that exhibitedby each of these compounds alone.

Also, in so far as the biocide composition of the invention according tofurther embodiments of the invention contains, in addition to thetwo-component combination of 2-methylisothiazolin-3-one and1,2-benzisothiazolin-3-one, one of the further active biocidalsubstances 3-iodo-2-propinyl-N-butyl carbamate,2-n-octylisothiazolin-3-one, formaldehyde or formaldehyde sourcematerial, or 2-bromo-2-nitropropane-1,3-diol, a synergistic biocidalactivity is achieved greater than that exhibited by the above-mentionedtwo-component combination and each of these further active substancesalone.

When the two-component combination is used together with one of theabove-mentioned further active biocidal substances, it contains2-methylisothiazolin-3-one and 1,2-benzisothiazolin-3-one, preferably ina weight ratio of 1:1. Any other weight ratio can also be selected,however, in so far as a synergistic activity is achieved thereby.

The biocide composition of the invention can be used in very differentfields. It is suitable, for example, for use in paints, plasters,lignosulfonates, chalk suspensions, adhesives, photochemicals,casein-containing products, starch-containing products, bituminousemulsions, surfactant solutions, motor fuels, cleaning agents, cosmeticproducts, water circulating systems, polymer dispersions, and coolinglubricants, against attack by, for example, bacteria, filamentous fungi,yeasts, and algae.

In practice, the biocide composition can be used either as aready-to-use mixture or by adding the biocides and the remainingcomponents of the composition separately to the substance to bepreserved.

The examples illustrate the invention.

In all the examples in which an active substance mixture of MIT and BITas well as additionally a further active biocidal substance was used,the weight ratio of MIT to BIT was 1:1.

EXAMPLE 1

This example shows the synergy of the two essential active substances inthe biocide composition of the invention.

For this purpose, aqueous mixtures were produced with variousconcentrations of 2-methylisothiazolin-3-one (MIT) and1,2-benzisothiazolin-3-one (BIT) and the activity of these mixtures onEscherichia coli (International Mycological Institute, Strain No. IMI362054) was tested.

In addition to the biocide component and water, the aqueous mixturescontained a nutrient medium, namely a Müller-Hinton broth (commercialproduct “Merck No. 10393”). The cell density of Escherichia coli was 10⁶cells/mL. The incubation time was 72 h at 25° C. Each sample wasincubated on an incubation shaker at 120 rpm.

Table 1 below shows the concentrations of MIT and BIT used. The tablealso shows whether growth of the microorganism took place (symbol “+”)or not (symbol “−”).

Thus, table 1 also shows the minimum inhibition concentrations (MIC).According to this, an MIC value of 17.5 ppm is found when MIT is usedalone and an MIC value of 25 ppm when BIT is used alone. In contrast,the MIC values of mixtures of MIT and BIT are clearly lower, i.e., whenthey are combined, MIT and BIT act synergistically.

TABLE I MIC values for Escherichia coli at an incubation time of 72 hMIT concentration BIT concentration (ppm) (ppm) 35 30 25 20 17.5 15 12.510 7.5 5 2.5 1 0 25 − − − − − − − − − − − − − 17.5 − − − − − − − − − − −− − 15 − − − − − − − − − − − − + 12.5 − − − − − − − − − − − + + 10 − − −− − − − − − − − + + 7.5 − − − − − − − − − + + + + 5 − − − − − −− + + + + + + 2.5 − − − − + + + + + + + + + 1 − − − + + + + + + + + + +0 − − − + + + + + + + + + +

The synergy that occurs is represented numerically by means of thecalculation of the synergy index given in Table II. The synergy index iscalculated according to the method of F. C. Kull et al., AppliedMicrobiology, Vol. 9 (1961), p. 538. Here the synergy index iscalculated using the following formula:

 Synergy index SI=Q _(a) /Q _(A) +Q _(b) /Q _(B).

When this formula is used on the biocide system tested here, thequantities in the formula have the following meaning:

Q_(a)=concentration of BIT in the biocide mixture of BIT and MIT

Q_(A)=concentration of BIT as sole biocide

Q_(b)=concentration of MIT in the biocide mixture of MIT and BIT

Q_(B)=concentration of MIT as sole biocide

When the synergy index exhibits a value greater than 1, this means thatthere is an antagonism. When the synergy index has the value 1, thismeans that there is an addition of the activity of the two biocides.When the synergy index has a value of less than 1, this means that asynergy of the two biocides exists.

TABLE II Calculation of the synergy index for Escherichia coli at anincubation time of 72 h Total MIC at concentration BIT MIT BIT + MITConcentration concentration concentration Q_(a) + Q_(b) BIT MIT Synergyindex Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt) Q_(a)/Q_(A)Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 17.5 17.5 0.0 100.0 0.00 1.001.00 1 15 16 6.3 93.8 0.04 0.86 0.90 2.5 10 12.5 20.0 80.0 0.10 0.570.67 7.5 7.5 15 50.0 50.0 0.30 0.43 0.73 12.5 5 17.5 71.4 28.6 0.50 0.290.79 20 2.5 22.5 88.9 11.1 0.80 0.14 0.94 25 0 25 100.0 0.0 1.00 0.001.00

It can be seen from Table II that the optimum synergy, i.e., the lowestsynergy index (0.67) of an MIT/BIT mixture, is at a mixture of 80% by wtof MIT and 20% by wt of BIT.

EXAMPLE 2

The synergy of the two active substances MIT and BIT against themicroorganism Pseudomonas putida is shown as in Example 1.

The test batches again contained a Müller-Hinton broth as a nutrientmedium. The cell density was 10⁶ cells/mL. The incubation time was 48 hat 25° C. Each sample was incubated on an incubation shaker at 120 rpm.

Table III below shows the MIC values of the tested biocide compositions.The MIC value was 12.5 ppm when MIT alone was used, and 60 ppm when BITalone was used.

TABLE III MIC values for Pseudomonas putida at an incubation time of 48h MIT MIT concentration BIT concentration (ppm) (ppm) 80 70 60 50 40 3025 20 15 10 7.5 5 2.5 1 0.5 0 17.5 − − − − − − + − − − − − − − − − 12.5− − − − − − − − − − − − − − − − 10 − − − − − − − − − − − − − + + + 7.5 −− − − − − − − − − − − + + + + 5 − − − − − − − − − + + + + + + + 2.5 − −− − − − − − + + + + + + + + 1 − − − − − − − + + + + + + + + + 0.5 − − −− − + + + + + + + + + + + 0 − − − + + + + + + + + + + + + +

With the simultaneous use of MIT and BIT a synergy occurred. Table IVshows the calculation of the synergy index. According to this, forPseudomonas putida the lowest synergy index (0.50) was at a mixture of3.8% by wt of MIT and 96.2% by wt of BIT.

TABLE IV Calculation of the synergy index for Pseudomonas putida at anincubation time of 48 h Total MIC at concentration BIT MIT BIT + MITConcentration concentration concentration Q_(a) + Q_(b) BIT MIT Synergyindex Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt) Q_(a)/Q_(A)Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 12.5 12.5 0.0 100.0 0.00 1.001.00 2.5 10 12.5 20.0 80.0 0.04 0.80 0.84 5 7.5 12.5 40.0 60.0 0.08 0.600.68 15 5 20 75.0 25.0 0.25 0.40 0.65 20 2.5 22.5 88.9 11.1 0.33 0.200.53 25 1 26 96.2 3.8 0.42 0.08 0.50 40 0.5 40.5 98.8 1.2 0.67 0.04 0.7160 0 60 100.0 0.0 1.00 0.00 1.00

EXAMPLE 3

The synergy of MIT and BIT against the microorganism Pseudomonasstutzeri is shown as in Example 1.

The test batches again contained a Müller-Hinton broth as a nutrientmedium. The cell density was 10⁶ cells/mL. The incubation time was 72 hat 25° C. Each sample was incubated on an incubation shaker at 120 rpm.

Table V below shows the MIC values of the tested biocide compositions.The MIC value was 12.5 ppm when MIT alone was used, and 20 ppm when BITalone was used.

TABLE V MIC values for Pseudomonas stutzeri at an incubation time of 72h MIT concentration BIT concentration (ppm) (ppm) 30 25 20 15 10 7.5 52.5 1 0.5 0 30 25 − − − − − − − − − − − 20 − − − − − − − − − − − 17.5 −− − − − − − − − − − 15 − − − − − − − − − − − 12.5 − − − − − − − − − − −10 − − − − − − − − − − + 7.5 − − − − − − − + + + + 5 − − − − − −− + + + + 2.5 − − − − − + + + + + + 1 − − − − + + + + + + + 0.5 − −− + + + + + + + + 0 − − − + + + + + + + +

With the simultaneous use of MIT and BIT, a synergy occurred. Table VIshows the calculation of the synergy index. According to this, thelowest synergy index (0.65) for Pseudomonas stutzeri was at a mixture of50% by wt of MIT and 50% by wt of BIT.

TABLE VI Calculation of the synergy index for Pseudomonas stutzeri at anincubation time of 72 h Total MIC at concentration BIT MIT BIT + MITConcentration concentration concentration Q_(a) + Q_(b) BIT MIT Synergyindex Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt) Q_(a)/Q_(A)Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 12.5 12.5 0.0 100.0 0.00 1.001.00 0.5 10 10.5 4.8 95.2 0.03 0.80 0.83 5 5 10 50.0 50.0 0.25 0.40 0.6510 2.5 12.5 80.0 20.0 0.50 0.20 0.70 15 1 16 93.8 6.3 0.75 0.08 0.83 200 20 100.0 0.0 1.00 0.00 1.00

EXAMPLE 4

The synergy of the two active substances MIT and BIT against themicroorganism Klebsiella pneumoniae is shown as in example 1.

The test batches again contained a Müller-Hinton broth as a nutrientmedium. The cell density was 10⁶ cells/mL. The incubation time was 72 hat 25° C. Each sample was incubated on an incubation shaker at 120 rpm.

Table VII below shows the MIC values of the tested biocide compositions.The MIC value was 20 ppm when MIT alone was used, and 25 ppm when BITalone was used.

TABLE VII MIC values for Klebsiella pneumoniae at an incubation time of72 h MIT concentration BIT concentration (ppm) (ppm) 35 30 25 20 15 107.5 5 2.5 1 0 25 − − − − − − − − − − − 20 − − − − − − − − − − − 17.5 − −− − − − − − − + + 15 − − − − − − − − − + + 12.5 − − − − − − − − − + + 10− − − − − − − − + + + 7.5 − − − − − − − + + + + 5 − − − − − + + + + + +2.5 − − − + + + + + + + + 1 − − − + + + + + + + + 0 − − − + + + + + + ++

With the simultaneous use of MIT and BIT, a synergy occurred. Table VIIIshows the calculation of the synergy index. According to this, thelowest synergy index (0.68) for Pseudomonas aeruginosa [sic] was at amixture of 50% by wt of MIT and 50% by wt of BIT.

TABLE VIII Calculation of the synergy index for Klebsiella pneumoniae atan incubation time of 72 h Total MIC at concentration BIT MIT BIT + MITConcentration concentration concentration Q_(a) + Q_(b) BIT MIT Synergyindex Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt) Q_(a)/Q_(A)Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 20 20 0.0 100.0 0.00 1.00 1.002.5 12.5 15 16.7 83.3 0.10 0.63 0.73 5 10 15 33.3 66.7 0.20 0.50 0.707.5 7.5 15 50.0 50.0 0.30 0338 0.68 15 5 20 75.0 25.0 0.60 0.25 0.85 250 25 100.0 0.0 1.00 0.00 1.00

EXAMPLE 5

The synergy of the two active substances MIT and BIT against themicroorganism Pseudomonas aeruginosa is shown as in Example 1.

The test batches again contained a Müller-Hinton broth as a nutrientmedium. The cell density was 10⁶ cells/mL. The incubation time was 48 hat 25° C. Each sample was incubated on an incubation shaker at 120 rpm.

Table IX below shows the MIC values of the tested biocide compositions.The MIC value was 30 ppm when MIT alone was used, and 150 ppm when BITalone was used.

TABLE IX MIC values for Pseudomonas aeruginosa at an incubation time of48 h MIT concentration BIT concentration (ppm) (ppm) 200 175 150 125 10075 50 25 10 5 0 50 − − − − − − − − − − − 40 − − − − − − − − − − − 30 − −− − − − − − − − − 20 − − − − − − − − − + + 10 − − − − − − − + + + + 5 −− − − − + + + + + + 2.5 − − − − + + + + + + + 1 − − − + + + + + + + + 0− − − + + + + + + + +

With the simultaneous use of MIT and BIT, a synergy occurred. Table Xshows the calculation of the synergy index. According to this, thelowest synergy index (0.67) for Pseudomonas aeruginosa was at a mixtureof 16.7% by wt of MIT and 83.3% by wt of BIT.

TABLE X Calculation of the synergy index for Pseudomonas aeruginosa atan incubation time of 48 h Total MIC at concentration BIT MIT BIT + MITConcentration concentration concentration Q_(a) + Q_(b) BIT MIT Synergyindex Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt) Q_(a)/Q_(A)Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 30 30 0.0 100.0 0.00 1.00 1.0010 20 30 33.3 66.7 0.07 0.67 0.73 50 10 60 83.3 16.7 0.33 0.33 0.67 1005 105 95.2 4.8 0.67 0.17 0.83 125 2.5 127.5 98.0 2.0 0.83 0.08 0.92 1500 150 100.0 0.0 1.00 0.00 1.00

EXAMPLE 6

A biocide composition of the following components was produced:Component Amount (% by wt) 2-Methylisothiazolin-3-one 5.1 (98% by wt)1,2-Benzisothiazolin-3-one 6.7 (in the form of a mixture of 74.6% by wtof BIT and 25.4% by wt of water; commercial product “Acticide ® BIT” ofthe company Thor Chemie GmbH) Polyethylene glycol 88.2 (average molarweight 400 g/mol) 100.0

The ready-to-use formulation of the biocide composition is a clearsolution, which can be attributed to the polyethylene glycol. Thesolution is suitable for use, e.g., in paints, polymer dispersions,plaster systems, and cooling lubricants, against attack by bacteria,filamentous fungi and yeasts.

EXAMPLE 7

A biocide composition of the following components was produced:

Component Amount (% by wt) 2-Methylisothiazolin-3-one 25 (in the form ofa solution of 20% by wt of MIT in water) 1,2-Benzisothiazolin-3-one 25(in the form of a suspension of 20% by wt of BIT in water; commercialproduct “Acticide ® BW 20” of the company Thor Chemie GmbH) Thickenerbased on xanthan 0.4 (commercial product “Rhodopol 50 MD” of the companyRhône-Poulenc) Defoaming agent 0.1 (commercial product “Drewplus T 4202”of the company Drew Ameroid) Water 49.5 100.0

In the ready-to-use formulation of the biocide composition, the BIT ispresent in finely suspended form. The formulation is suitable for theapplications given in the specification above.

EXAMPLE 8

A biocide composition of the following components was produced:

Component Amount (% by wt) 1,2-Benzisothiazolin-3-one 3.35 (as a mixtureof 74.6% by wt of BIT and 25.4% by wt of water) Water 92.8 Sodiumhydroxide solution 1.3 (50% by wt in water) 2-Methylisothiazolin-3-one2.55 (98% by wt) 100.0

The additional water was added to the BIT/water mixture (weight ratio74.6:25.4) and then the mixture was converted into a solution by addingthe sodium hydroxide solution, which is formed owing to the formation ofthe corresponding sodium salt. Finally the MIT was added. Theready-to-use formulation was a clear solution and had a pH value ofabout 8.2.

The ready-to-use formulation of the biocide composition is suitable forthe use applications given above.

EXAMPLE 9

Biocide compositions according to the invention were incorporated into acoating composition A that is used for coating building facades. Thiscoating composition is a plaster based on an aqueous polymer dispersioncommercially available under the designation “Granol KR 3.0” (companySteinwerke Kupferdreh GmbH). The addition of the biocide composition ofthe invention served to preserve the coating composition before its use,i.e., while it was stored in its packing drums.

The biocides shown in Table XI below were added respectively to 50 g ofcoating composition A. The stated amounts of biocide refer to the amountof coating composition A. In the MIT/BIT mixtures, the two biocides werepresent in a weight ratio of 1:1.

Apart from a blank test without addition of biocide, 1 mL of a standardbacterial inoculum that contained the following bacterial strains wasadded to each sample of coating composition A:

Shewanella putrefaciens

Alcaligenes faecalis

Serratia liquefaciens

Klebsiella species

Proteus penneri/vulgaris

Providencia rettgeri

Pseudomonas fluorescens

Pseudomonas aeruginosa

Pseudomonas stutzeri

Escherichia coli

Corynebacterium pseudodiphteriae

Cellulomonas flavigena

Corynebacterium species

The cell density of the inoculum was 10¹⁰ to 3·10¹⁰ cells/mL, and thecell density of the samples was 2·10⁸ to 6·10⁸ cells/g. The samples wereheld at 30° C. for 7 days. Then a streak of each sample was produced ona nutrient agar plate, held at 30° C. for 48 h, and then evaluated forbacterial growth. The following evaluation scale was used:

0=no growth

1=minimal growth up to 10 colonies

2=slight growth up to 100 colonies

3=moderate growth up to 300 colonies

4=uniform growth, single colonies still discernible

5 =strong growth, too many colonies to count, but not covering entiresurface

6=extensive growth, almost no individual colonies, total streak surfaceovergrown

When the bacterial growth was evaluated as below 6, a second bacterialinoculum of the above-mentioned type was added to the correspondingoriginal 50 g sample and it was again held at 30° C. for 7 days. Then astreak was again produced on a nutrient agar plate, which was againevaluated for its bacterial growth after a holding time of 48 h at 30°C.

When the bacterial growth of the streak was evaluated as 6 for a sample,the testing of this sample was terminated. As long as a sample had notreached this number, a bacterial inoculum was again added in theabove-mentioned manner and the sample was held and tested by means of astreak. This procedure was repeated as necessary, with a maximum of 4bacterial inocula being added per sample.

Table XI summarizes the results for coating composition A.

TABLE XI Coating composition A Biocide Bacterial growth (% by wt) (7days + 48 h after 4^(th) inoculation) none (Growth already after 1^(st)inoculation) BIT 0.005 (Growth already after 1^(st) inoculation) 0.01(Growth already after 1^(st) inoculation) 0.015 (Growth already after2^(nd) inoculation) 0.02 (Growth already after 2^(nd) inoculation) 0.03(Growth already after 3^(rd) inoculation) MIT 0.005 6 0.01 5 0.015 50.02 4 0.03 0 MIT/BIT 0.005 5 0.01 0 0.015 0 0.02 0 0.03 0

It can be seen from Table XI that the sample without addition of biocidedeveloped full bacterial growth already after the first inoculum.

When BIT alone was added, full bacterial growth was reached alreadyafter the first inoculation at 0.005% by wt of BIT, after the secondinoculation at 0.015% by wt of BIT, and after the third inoculation at0.03% by wt of BIT.

When MIT alone was added, full bacterial growth was reached only afterfour inoculations, in fact at the smallest amount of biocide of 0.005%by wt. But a uniform to strong bacterial growth was still found also atthe higher biocide concentrations of 0.01, 0.015, and 0.02% by wt ofMIT. Only in a sample with the highest concentration of 0.03% by wt ofMIT was no bacterial growth found even after four inoculations.

In contrast, the biocide composition of the invention of MIT and BITproved itself to be considerably more effective. After fourinoculations, a distinct bacterial growth occurred only at the lowestconcentration of 0.005% by wt of MIT/BIT. Bacterial growth in coatingcomposition A was prevented completely at the higher concentrations inthe range of 0.01 to 0.03% by wt of MIT/BIT.

EXAMPLE 10

Example 9 was repeated, but using coating composition B instead ofcoating composition A.

Coating composition B is a particularly low-emission plaster based on apolymer dispersion commercially available under the designation “GranolKR 3.0 LF” (company Steinwerke Kupferdreh GmbH).

The results using coating composition B are summarized in Table XIIbelow.

TABLE XII Coating composition B Biocide Bacterial growth (% by wt) (7days + 48 h after 4^(th) inoculation) none (Growth already after 1^(st)inoculation) BIT 0.005 (Growth already after 1^(st) inoculation) 0.01(Growth already after #^(st) inoculation) 0.015 (Growth already after2^(nd) inoculation) 0.02 (Growth already after 2^(nd) inoculation) 0.03(Growth already after 3^(rd) inoculation) MIT 0.005 4 0.01 1 0.015 10.02 0 0.03 0 MIT/BIT 0.005 5 0.01 0 0.015 0 0.02 0 0.03 0

The results for coating composition B are largely in agreement withthose for coating composition A.

Full bacterial growth also took place already after the firstinoculation in the case of coating composition B.

When BIT alone was used, complete bacterial attack could be observedafter the third inoculation at the latest.

When MIT alone was used, bacterial growth could be prevented completelyafter the fourth inoculation only with the highest concentrations of0.02 and 0.03% by wt.

In contrast, it was possible to suppress bacterial growth completelywith the MIT/BIT combination according to the invention even at therelatively low concentration of 0.01% by wt.

EXAMPLE 11

The synergy of the two active substances MIT and BIT against themicroorganism Aspergillus niger is shown as in Example 1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The spore concentration was 10⁶ per mL. The incubation time was96 h at 25° C. Each sample was incubated on an incubation shaker at 120rpm.

Table XIII below shows the MIC values of the tested biocidecompositions. The MIC value was 750 ppm when MIT alone was used, and 100ppm when BIT alone was used.

TABLE XIII MIC values for Aspergillus niger at an incubation time of 96h MIT concentration BIT concentration (ppm) (ppm) 150 100 75 50 25 10 50 750 − − − − − − − − 500 − − − − − − + + 250 − − − − − + + + 100 − − −− + + + +  75 − − − − + + + +  50 − − − + + + + +  25 − − − + + + + + 10 − − + + + + + +  0 − − + + + + + +

With the simultaneous use of MIT and BIT, a synergy occurred. Table XIVshows the calculation of the synergy index. According to this, thelowest synergy index (0.57) for Aspergillus niger was at a mixture of50% by wt of MIT and 50% by wt of BIT.

TABLE XIV Calculation of the synergy index for Aspergillus niger at anincubation time of 96 h Total MIC at concentration BIT MIT BIT + MITConcentration concentration concentration Q_(a) + Q_(b) BIT MIT Synergyindex Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt) Q_(a)/Q_(A)Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 100 100 0.0 100.0 0.00 1.00 1.0025 75 100 25.0 75.0 0.03 0.75 0.78 50 75 125 40.0 60.0 0.07 0.75 0.82 5050 100 50.0 50.0 0.07 0.50 0.57 75 75 150 50.0 50.0 0.10 0.75 0.85 75 50125 60.0 40.0 0.10 0.50 0.60 100 50 150 66.7 33.3 0.13 0.50 0.63 250 50300 83.3 16.7 0.33 0.50 0.83 250 25 275 90.9 9.1 0.33 0.25 0.58 500 25525 95.2 4.8 0.67 0.25 0.92 500 10 510 98.0 2.0 0.67 0.50 0.77 750 0 750100.0 0.0 1.00 0.00 1.00

EXAMPLE 12

The synergy of the two active substances MIT and BIT against themicroorganism Penicillium funiculosum is shown as in Example 1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The spore concentration was 10⁶ per mL. The incubation time was96 h at 25° C. Each sample was incubated on an incubation shaker at 120rpm.

Table XV below shows the MIC values of the tested biocide compositions.The MIC value was 200 ppm when MIT alone was used, and 40 ppm when BITalone was used.

TABLE XV MIC values for Penicillium funculosum at an incubation time of96 h MIT concentration BIT concentration (ppm) (ppm) 75 50 40 30 20 1510 0 200 − − − − − − − − 150 − − − − − − − + 100 − − − − − − − +  75 − −− − − − + +  50 − − − − − + + +  25 − − − − − + + +  10 − − − − − + + + 5 − − − + + + + +  0 − − − + + + + +

With the simultaneous use of MIT and BIT, a synergy occurred. Table XVIshows the calculation of the synergy index. According to this, thelowest synergy index (0.55) for Penicillium funiculosum was at a mixtureof 33.3% by wt of MIT and 66.7% by wt of BIT.

TABLE XVI Calculation of the synergy index for Penicillium funiculosumat an incubation time of 96 h Total MIC at concentration BIT MIT BIT +MIT Concentration concentration concentration Q_(a) + Q_(b) BIT MITSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 40 40 0.0 100.0 0.001.00 1.00 10 30 40 25.0 75.0 0.05 0.75 0.80 10 20 30 33.3 66.7 0.05 0.500.55 25 30 55 45.5 54.5 0.13 0.75 0.88 25 20 45 55.6 44.4 0.13 0.50 0.6350 20 70 71.4 28.6 0.25 0.50 0.75 75 20 95 78.9 21.1 0.38 0.50 0.88 7515 90 83.3 16.7 0.38 0.38 0.75 100 15 115 87.0 13.0 0.50 0.38 0.88 10010 110 90.9 9.1 0.50 0.25 0.75 200 0 200 100.0 0.0 1.00 0.00 1.00

EXAMPLE 13

The synergy of an active substance mixture that contains3-iodo-2-propinyl-N-butyl carbamate (IPBC) in addition to MIT and BIT,against the microorganism Aspergillus niger is shown as in Example 1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The spore concentration was 10⁶ per mL. The incubation time was72 h at 25° C. Each sample was incubated on an incubation shaker at 120rpm.

Table XVII below shows the MIC values of the tested biocidecompositions. The MIC value was 150 ppm when MIT/BIT alone was used, and2.5 ppm when IPBC alone was used.

TABLE XVII MIC values for Aspergillus niger at an incubation time of 72h MIT/BIT concentration IPBC concentration (ppm) (ppm) 5 4.5 4 3.5 3 2.52 1.5 1 0.75 0 250 − − − − − − − − − − − 225 − − − − − − − − − − − 200 −− − − − − − − − − − 175 − − − − − − − − − − − 150 − − − − − − − − − − −125 − − − − − − − − − − + 100 − − − − − − − − − − + 75 − − − − − − − − −− + 50 − − − − − − − − + + + 25 − − − − − − + + + + + 10 − − − − −− + + + + + 0 − − − − − − + + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of IPBC, a synergy occurred. Table XVIII shows the calculationof the synergy index. According to this, the lowest synergy index (0.80)for Aspergillus niger was at a mixture of, on the one hand, 99.0% by wtof MIT/BIT and, on the other hand, 1.0% by wt of IPBC.

TABLE XVIII Calculation of the synergy index for Aspergillus niger at anincubation time of 72 h Total MIC at concentration MIT/BIT IPBCBIT/MIT + IPBC Concentration concentration concentration Q_(a) + Q_(b)MIT/BIT IPBC Synergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% bywt) Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 150 0 150 100.00.0 1.00 0.00 1.00 75 0.75 75.75 99.0 1.0 0.50 0.30 0.80 75 1 76 98,71.3 0.50 0.40 0.90 50 1.5 51.5 97.1 2.9 0.33 0.60 0.93 0 2.5 2.5 0.0100.0 0.00 1.00 1.00

EXAMPLE 14

The synergy of an active substance mixture that contains IPBC inaddition to MIT and BIT, against the microorganism Penicilliumfuniculosum is shown as in Example 1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The spore concentration was 10⁶ per mL. The incubation time was72 h at 25° C. Each sample was incubated on an incubation shaker at 120rpm.

Table XIX below shows the MIC values of the tested biocide compositions.The MIC value was 20 ppm when the MIT/BIT mixture alone was used, and0.75 ppm when IPBC alone was used.

TABLE XIX MIC values for Penicillium funiculosum at an incubation timeof 72 h MIT/BIT concentration IPBC concentration (ppm) (ppm) 2 1.75 1.51.25 1 0.75 0.5 0.3 0.2 0.1 0 50 − − − − − − − − − − − 45 − − − − − − −− − − − 40 − − − − − − − − − − − 35 − − − − − − − − − − − 30 − − − − − −− − − − − 25 − − − − − − − − − − − 20 − − − − − − − − − − − 15 − − − − −− − − − − + 12.5 − − − − − − − − − + + 10 − − − − − − − − − + + 7.5 − −− − − − − + + + + 0 − − − − − − + + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of IPBC, a synergy occurred. Table XX shows the calculation ofthe synergy index. According to this, the lowest synergy index (0.77)for Penicillium funiculosum was at a mixture of, on the one hand, 98.0%by wt of MIT/BIT and, on the other hand, 2.0% by wt of IPBC.

TABLE XX Calculation of the synergy index for Penicillium funiculosum atan incubation time of 72 h MIC at Total MIT/BIT IPBC concentrationConcentration concentration concentration MIT/BIT + IPBC MIT/BIT IPBCSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 0.75 0.75 0.0 100.00.00 1.00 1.00 10 0.3 10.3 97.1 2.9 0.50 0.40 0.90 10 0.2 10.2 98.0 2.00.50 0.27 0.77 12.5 0.2 12.7 98.4 1.6 0.63 0.27 0.89 15 0.1 15.1 99.30.7 0.75 0.13 0.88 20 0 20 100.0 0.0 1.00 0.00 1.00

EXAMPLE 15

The synergy of an active substance mixture that contains2-n-octylisothiazolin-3-one (OIT) in addition to MIT and BIT, againstthe microorganism Aspergillus niger is shown as in Example 1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The spore concentration was 10⁶ per mL. The incubation time was72 h at 25° C. Each sample was incubated on an incubation shaker at 120rpm.

Table XXI below shows the MIC values of the tested biocide compositions.The MIC value was 100 ppm when MIT/BIT alone was used, and 5 ppm whenOIT alone was used.

TABLE XXI MIC values for Aspergillus niger at an incubation time of 72 hMIT/BIT concentration OIT concentration (ppm) (ppm) 10 7.5 5 2.5 1 0.50.25 0 200 − − − − − − − − 150 − − − − − − − − 100 − − − − − − − −  80 −− − − − − − +  60 − − − − − + + +  40 − − − − + + + +  30 − − −− + + + +  20 − − − + + + + +  10 − − − + + + + +  0 − − − + + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of OIT, a synergy occurred. Table XXII shows the calculation ofthe synergy index. According to this, the lowest synergy index (0.80)for Aspergillus niger was at a mixture of, on the one hand, 92.3% by wtof MIT/BIT and, on the other hand, 7.7% by wt of OIT, as well as at amixture, of on the one hand, 98.4% by wt of MIT/BIT and, on the otherhand, 1.6% by wt of OIT.

TABLE XXII Calculation of the synergy index for Aspergillus niger at anincubation time of 72 h MIC at Total MIT/BIT OIT concentrationConcentration concentration concentration MIT/BIT + OIT MIT/BIT OITSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 5 5 0.0 100.0 0.001.00 1.00 30 2.5 32.5 92.3 7.7 0.30 0.50 0.80 40 2.5 42.5 94.1 5.9 0.400.50 0.90 60 1 61 98.4 1.6 0.60 0.20 0.80 80 0.5 80.5 99.4 0.6 0.80 0.100.90 100 0 100 100.0 0.0 1.00 0.00 1.00

EXAMPLE 16

The synergy of an active substance mixture that contains OIT in additionto MIT and BIT, against the microorganism Penicillium funiculosum isshown as in Example 1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The spore concentration was 10⁶ per mL. The incubation time was72 h at 25° C. Each sample was incubated on an incubation shaker at 120rpm.

Table XXIII below shows the MIC values of the tested biocidecompositions. The MIC value was 50 ppm when MIT/BIT alone was used, and5 ppm when OIT alone was used.

TABLE XXIII MIC values for Penicillium funiculosum at an incubation timeof 72 h MIT/BIT concentration OIT concentration (ppm) (ppm) 5 2.5 1 0.50.25 0 75 − − − − − − 50 − − − − − − 25 − − − − − + 15 − − − + + + 10 −− + + + +  5 − − + + + +  0 − + + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of OIT, a synergy occurred. Table XXIV shows the calculation ofthe synergy index. According to this, the lowest synergy index (0.50)for Penicillium funiculosum was at a mixture of 93.8% by wt of MIT/BIT,on the one hand, and 6.2% by wt of OIT, on the other hand.

TABLE XXIV Calculation of the synergy index for Penicillium funiculosumat an incubation time of 72 h MIC at Total MIT/BIT OIT concentrationConcentration concentration concentration MIT/BIT + OIT MIT/BIT OITSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 5 5 0.0 100.0 0.001.00 1.00 5 2.5 7.5 66.7 33.3 0.10 0.50 0.60 10 2.5 12.5 80.0 20.0 0.200.50 0.70 15 2.5 17.5 85.7 14.3 0.30 0.50 0.80 15 1 16 93.8 6.2 0.300.20 0.50 25 1 26 96.2 3.8 0.50 0.20 0.70 25 0.5 25.5 98.0 2.0 0.50 0.100.60 25 0.25 25.25 99.0 1.0 0.50 0.05 0.55 50 0 50 100.0 0.0 1.00 0.001.00

EXAMPLE 17

The synergy of an active substance mixture that contains OIT in additionto MIT and BIT, against the microorganism Saccharomyces cerevisiae isshown as in Example 1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The cell density was 10⁶ cells/mL. The incubation time was 72 hat 25° C. Each sample was incubated on an incubation shaker at 120 rpm.

Table XXV below shows the MIC values of the tested biocide compositions.The MIC value was 40 ppm when MIT/BIT alone was used, and 5 ppm when OITalone was used.

TABLE XXV MIC values for Saccharomyces cerevisiae at an incubation timeof 72 h MIT/BIT concentration OIT concentration (ppm) (ppm) 20 15 12.510 7.5 5 2.5 1 0.5 0.25 0 100 − − − − − − − − − − − 80 − − − − − − − − −− − 70 − − − − − − − − − − − 60 − − − − − − − − − − − 50 − − − − − − − −− − − 40 − − − − − − − − − − − 30 − − − − − − − − − − + 20 − − − − − −− + + + + 15 − − − − − − + + + + + 10 − − − − − − + + + + + 5 − − − − −− + + + + + 0 − − − − − − + + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of OIT, a synergy occurred. Table XXVI shows the calculation ofthe synergy index. According to this, the lowest synergy index (0.80)for Saccharomyces cerevisiae was at a mixture of 99.2% by wt of MIT/BITon the one hand and 0.8% by wt of OIT on the other hand.

TABLE XXVI Calculation of the synergy index for Saccharomyces cerevisiaeat an incubation time of 72 h MIC at Total MIT/BIT OIT concentrationConcentration concentration concentration MIT/BIT + OIT MIT/BIT OITSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 5 5 0.0 100.0 0.001.00 1.00 30 1 31 96.8 3.2 0.75 0.20 0.95 30 0.5 30.5 98.4 1.6 0.75 0.100.85 30 0.25 30.25 99.2 0.8 0.75 0.05 0.80 40 0 40 100.0 0.0 1.00 0.001.00

EXAMPLE 18

The synergy of an active substance mixture that contains OIT in additionto MIT and BIT, against the microorganism Pseudomonas aeruginosa isshown as in Example 1.

The test batches contained a Müller-Hinton broth as a nutrient medium.The cell density was 10⁶ cells/mL. The incubation time was 144 h at 25°C. Each sample was incubated on an incubation shaker at 120 rpm.

Table XXVII below shows the MIC values of the tested biocidecompositions. The MIC value was 30 ppm when MIT/BIT alone was used, andover 800 ppm when OIT alone was used.

TABLE XXVII MIC values for Pseudomonas aeruginosa at an incubation timeof 144 h MIT/BIT concentration OIT concentration (ppm) (ppm) 800 700 600500 400 300 200 100 75 50 25 10 7.5 5 0 75 − − − − − − − − − − − − − − −50 − − − − − − − − − − − − − − − 40 − − − − − − − − − − − − − − − 30 − −− − − − − − − − − − − − − 20 − − − − − − − − − − − + + + + 15 − − − − −− − + + + + + + + + 10 − − − − − + + + + + + + + + + 5 −− + + + + + + + + + + + + + 0 + + + + + + + + + + + + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of OIT, a synergy occurred. Table XXVIII shows the calculationof the synergy index. According to this, the lowest synergy index (0.53)for Pseudomonas aeruginosa was at a mixture of 44.4% by wt of MIT/BIT,on the one hand, and 55.6% by wt of OIT, on the other hand.

TABLE XXVIII Calculation of the synergy index for Pseudomonas aeruginosaat an incubation time of 144 h MIC at Total MIT/BIT OIT concentrationConcentration concentration concentration MIT/BIT + OIT MIT/BIT OITSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 900 900 0.0 100.00.00 1.00 1.00 5 700 705 0.7 99.3 0.13 0.78 0.90 10 600 610 1.6 98.40.25 0.67 0.92 10 500 510 2.0 98.0 0.25 0.56 0.81 10 400 410 2.4 97.60.25 0.44 0.69 15 300 315 4.8 95.2 0.38 0.33 0.71 15 200 215 7.0 93.00.38 0.22 0.60 20 100 120 16.7 83.3 0.50 0.11 0.61 20 75 95 21.1 78.90.50 0.08 0.58 20 50 70 28.6 71.4 0.50 0.06 0.56 20 25 45 44.4 55.6 0.500.03 0.53 40 0 40 100.0 0.0 1.00 0.00 1.00

EXAMPLE 19

The synergy of an active substance mixture that contains formaldehyde(HCHO) in addition to MIT and BIT, against the microorganism Escherichiacoli is shown as in Example 1.

The test batches contained a Müller-Hinton broth as a nutrient medium.The cell density was 10⁶ cells/mL. The incubation time was 48 b at 25°C. Each sample was incubated on an incubation shaker at 120 rpm.

Table XXIX below shows the MIC values of the tested biocidecompositions. The MIC value was 25 ppm when MIT/BIT alone was used, and300 ppm when HCHO alone was used.

TABLE XXIX MIC values for Escherichia coli at an incubation time of 48 hMIT/BIT concentration HCHO concentration (ppm) (ppm) 1000 900 800 700600 500 400 300 200 100 50 0 45 − − − − − − − − − − − − 40 − − − − − − −− − − − − 35 − − − − − − − − − − − − 30 − − − − − − − − − − − − 25 − − −− − − − − − − − − 20 − − − − − − − − − − − + 15 − − − − − − − − − − − +12.5 − − − − − − − − − − + + 10 − − − − − − − − − + + + 7.5 − − − − − −− − − + + + 5 − − − − − − − − + + + + 0 − − − − − − − − + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of HCHO, a synergy occurred. Table XXX shows the calculation ofthe synergy index. According to this, the lowest synergy index (0.77)for Escherichia coli was at a mixture of 23.1% by wt of MIT/BIT on theone hand and 76.9% by wt of HCHO on the other hand.

TABLE XXX Calculation of the synergy index for Escherichia coli at anincubation time of 48 h MIC at Total MIT/BIT HCHO concentrationConcentration concentration concentration MIT/BIT + HCHO MIT/BIT HCHOSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 300 300 0.0 100.00.00 1.00 1.00 7.5 200 207.5 3.6 96.4 0.30 0.67 0.97 12.5 100 112.5 11.188.9 0.50 0.33 0.83 15 50 65 23.1 76.9 0.60 0.17 0.77 25 0 25 100.0 0.01.00 0.00 1.00

EXAMPLE 20

The synergy of an active substance mixture that contains HCHO inaddition to MIT and BIT, against the microorganism Pseudomonasaeruginosa is shown as in Example 1.

The test batches contained a Müller-Hinton broth as a nutrient medium.The cell density was 10⁶ cells/mL. The incubation time was 48 h at 25°C. Each sample was incubated on an incubation shaker at 120 rpm.

Table XXXI below shows the MIC values of the tested biocidecompositions. The MIC value was 30 ppm when MIT/BIT alone was used, and300 ppm when HCHO alone was used.

TABLE XXXI MIC values for Pseudomonas aeruginosa at an incubation timeof 48 h MIT/BIT concentration HCHO concentration (ppm) (ppm) 1000 900800 700 600 500 400 300 200 100 50 0 45 − − − − − − − − − − − − 40 − − −− − − − − − − − − 35 − − − − − − − − − − − − 30 − − − − − − − − − − − −25 − − − − − − − − − − − + 20 − − − − − − − − − − − + 15 − − − − − − − −− − + + 12.5 − − − − − − − − − − + + 10 − − − − − − − − − + + + 7.5 − −− − − − − − − + + + 5 − − − − − − − − + + + + 0 − − − − − − − − + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of HCHO, a synergy occurred. Table XXXII shows the calculationof the synergy index. According to this, the lowest synergy index (0.75)for Pseudomonas aeruginosa was at a mixture of 11.1% by wt of MIT/BIT,on the one hand, and 88.9% by wt of HCHO, on the other hand.

TABLE XXXII Calculation of the synergy index for Pseudomonas aeruginosaat an incubation time of 48 h MIC at Total MIT/BIT HCHO concentrationConcentration concentration concentration MIT/BIT + HCHO MIT/BIT HCHOSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 0 300 300 0.0 100.00.00 1.00 1.00 7.5 200 207.5 3.6 96.4 0.25 0.67 0.92 12.5 100 112.5 11.188.9 0.42 0.33 0.75 15 100 115 13.0 87.0 0.50 0.33 0.83 20 50 70 28.671.4 0.67 0.17 0.83 30 0 30 100.0 0.0 1.00 0.00 1.00

EXAMPLE 21

The synergy of an active substance that contains2-bromo-2-nitropropane-1,3-diol (BNPD) in addition to MIT and BIT,against the microorganism Penicillium funiculosum is shown as in Example1.

The test batches contained a Sabouraud maltose broth as a nutrientmedium. The spore concentration was 10⁶ per mL. The incubation time was72 h at 25° C. Each sample was incubated on an incubation shaker at 120rpm.

Table XXXIII below shows the MIC values of the tested biocidecompositions. The MIC value was 25 ppm when MIT/BIT alone was used, and600 ppm when BNPD alone was used.

TABLE XXXIII MIC values for Penicillium funiculosum at an incubationtime of 72 h MIT/BIT concentration BNPD concentration (ppm) (ppm) 1000800 600 400 300 200 150 100 50 25 0 50 − − − − − − − − − − − 45 − − − −− − − − − − − 40 − − − − − − − − − − − 35 − − − − − − − − − − − 30 − − −− − − − − − − − 25 − − − − − − − − − − − 20 − − − − − − − − − − + 15 − −− − − − − − − + + 12.5 − − − − − − − − + + + 10 − − − − − − + + + + +7.5 − − − − − + + + + + + 0 − − − + + + + + + + +

When the above-mentioned mixture of MIT and BIT was used, as well as anaddition of BNPD, a synergy occurred. Table XXXIV shows the calculationof the synergy index. According to this, the lowest synergy index (0.67)for Penicillium funiculosum was at a mixture of, on the one hand, 11.1%by wt of MIT/BIT and, on the other hand, 88.9% by wt of BNPD.

TABLE XXXIV Calculation of the synergy index for Penicillium funiculosumat an incubation time of 72 h MIC at Total MIT/BIT BNPD concentrationConcentration concentration concentration MIT/BIT + BNPD MIT/BIT BNPDSynergy index Q_(a) (ppm) Q_(b) (ppm) (ppm) (% by wt) (% by wt)Q_(a)/Q_(A) Q_(b)/Q_(B) Q_(a)/Q_(A) + Q_(b)/Q_(B) 25 0 25 100.0 0.0 1.000.00 1.00 20 25 45 44.4 55.6 0.80 0.04 0.84 20 50 70 28.6 71.4 0.80 0.080.88 15 50 65 23.1 76.9 0.60 0.08 0.68 15 1oo 115 13.0 87.0 0.60 0.170.77 12.5 100 112.5 11.1 88.9 0.50 0.17 0.67 12.5 150 162.5 7.7 92.30.50 0.25 0.75 12.5 200 212.5 5.9 94.1 0.50 0.33 0.83 10 200 210 4.895.2 0.40 0.33 0.73 7.5 300 307.5 2.4 97.6 0.30 0.50 0.80 7.5 400 407.51.8 98.2 0.30 0.67 0.97 0 600 600 0.0 100.0 0.00 1.00 1.00

What is claimed is:
 1. A biocide composition comprising at least twoactive biocidal substances, selected from the group consisting of2-methylisothiazolin-3-one and 1,2-benzisothiazolin-3-one, said biocidecomposition being free of any 5-chloro-2-methylisothiazolin-3-one.
 2. Abiocide composition according to claim 1, wherein2-methylisothiazolin-3-one and 1,2-benzisothiazolin-3-one are present ina weight ratio of 50:1 to 1:50.
 3. A biocide composition according toclaim 2, wherein 2-methylisothiazolin-3-one and1,2-benzisothiazolin-3-one are present in a weight ratio of 15:1 to 1:8.4. A biocide composition as in any of the preceding claims, in whichsaid composition contains 2-methylisothiazolin-3-one and1,2-benzisothiazolin-3-one in a total concentration of 1 to 20% by wtrelative to the total biocide composition.
 5. A biocide composition asin claim 1, wherein said composition contains a polar and/or nonpolarliquid medium.
 6. A biocide composition according to claim 5, whereinsaid composition contains as a polar liquid medium, water, an aliphaticalcohol having 1 to 4 carbon atoms, a glycol, a glycol ether, a glycolester, a polyethylene glycol, a polypropylene glycol, N,N-dimethylformamide, or a mixture of such substances.
 7. A biocidecomposition according to claim 6, wherein the polar liquid medium iswater and said composition has a pH of 7 to
 9. 8. A biocide compositionaccording to claim 5, containing as a nonpolar liquid medium xyleneand/or toluene.
 9. A biocide composition according to claim 1,containing 3-iodo-2-propinyl-N-butyl carbamate as an active biocidalsubstance.
 10. A biocide composition according to claim 9, wherein theweight ratio of the combination of 2-methylisothiazolin-3-one and1,2-benzisothiazolin-3-one to 3-iodo-2-propinyl-N-butyl carbamate rangesfrom 1:10 to 100:1.
 11. A biocide composition according to claim 1containing 2-n-octylisothiazolin-3-one as an active biocidal substance.12. A biocide composition according to claim 11, wherein the weightratio of the combination of 2-methylisothiazolin-3-one and1,2-benzisothiazolin-3-one to 2-n-octylisothiazolin-3-one ranges from1:10 to 100:1.
 13. A biocide composition according to claim 1 containingformaldehyde or a formaldehyde source material as an active biocidalsubstance.
 14. A biocide composition according to claim 13, wherein theweight ratio of the combination of 2-methylisothiazolin-3-one and1,2-benzisothiazolin-3-one to the formaldehyde source material rangesfrom 1:100 to 10:1.
 15. A biocide composition according to claim 1additionally containing 2-bromo-2-nitropropane-1,3-diol as an activebiocidal substance.
 16. A biocide composition according to claim 15,wherein the weight ratio of the combination of2-methylisothiazolin-3-one and 1,2-benzisothiazolin-3-one to2-bromo-2-nitropropane-1,3-diol ranges from 1:10 to 10:1.
 17. A biocidecomposition according to claim 9 containing 2-methylisothiazolin-3-oneand 1,2-benzisothiazolin-3-one in a weight ratio of 1:1.
 18. A methodfor controlling harmful microorganisms which comprises applying to saidharmful microorganisms a biocide composition, said compositioncomprising: 1) 2-methylisothiazolin-3-one; and 2)1,2-benzisothiazolin-3-one and; 3) said biocide composition being freeof 5-chloro-2-methylisothiazolin-3-one.